The fundamental interest of the Myeloid Malignancies Section is the detection, prevention and treatment of acute myeloid leukemia (AML) relapse with the long-term objective of using immunotherapy without the need for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Foundational to this objective is the development of high sensitivity biomarkers for residual AML in those patients who have been treated to remission but remain at risk of relapse. Work in our laboratory from previous years had determined, using customized medium-throughput quantitative real-time PCR (RQ-PCR) arrays, the gene expression profiles of a wide range of ex-vivo primary samples from untreated AML patients and healthy controls. This allowed us to develop a multi-gene expression panel for AML measurable residual disease (MRD) where any level of gene expression above the level seen in healthy controls was considered abnormal. This five-gene MG-MRD panel (WT1, PRAME, PRTN3, CCNA1, MSLN) was retrospectively tested on 74 patients who underwent allo-HSCT at the NHLBI in the period 1994-2012. Testing of peripheral blood samples prior to transplantation demonstrated excellent concordance with traditional bone marrow based evaluation and improved risk stratification for post transplant relapse and overall survival outcomes. Pre-SCT assessment by MG-MRD predicted all clinical relapses occurring in the first 100 days after allogeneic hematopoietic stem cell transplantation compared with 57% sensitivity using the prior research standard MRD RQ-PCR test (ie: WT1 alone). Nine patients who were negative for WT1 prior to transplantation were correctly reclassified into a high-risk MG-MRD-positive group, associated with 100% post-transplant mortality. This study provided proof of principle that a multiple gene approach may be superior to the use of WT1 expression alone for AML residual disease detection. Status: Published in Bone Marrow Transplantation, May 2015 (PMID: 25665046). This MG-MRD testing is now being used as an AML MRD biomarker in collaboration with investigators performing AML clinical trials at Johns Hopkins, Duke, University of North Carolina, University of California San Francisco and the University of Chicago, including serving as the laboratory center for this correlate in two ongoing multi-center NCI Cancer Therapy Evaluation Program (CTEP) sponsored clinical trials of novel immunotherapy approaches for patients with AML. In addition, we have, in collaboration with Brent Wood and Jerry Radich in Seattle (who performed flow cytometric based MRD testing) and Gabriel Mannis, Aaron Logan and Tom Martin (all UCSF), recently attempted to correlate measurable residual disease in the graft product with post-transplant relapse outcomes in what we believe is the largest retrospective cohort of adult AML autologous hematopoietic stem cell transplantation patients assessed for MRD (72 patients transplanted at UCSF in the period 2004-2013). This work represented an unique technical challenge and necessitated the development of several additional assays in our laboratory (Inv 16, t(8,21) and t(15:17) specific PCRs and also NPM1 type A, B and D mutation PCRs) to add to our standard expression targets. This multi-multi-gene (MMG) panel was able to substantially outperform both flow cytometry and conventional research PCR based AML MRD testing (WT1 alone). Status: Manuscript in preparation. The focus of the Myeloid Malignancies Section in the initial establishment phase was predominately on translational laboratory research on retrospectively collected, clinical annotated, samples. Towards the end of this period however we were given the mandate to expand into prospective clinical investigation of patients with myeloid malignancies at the NIH Clinical Center. A staff clinician has been recruited to help establish a weekly outpatient clinic and serve as clinical lead on new section protocols. In addition to protocols J1293 (Characterization of the immune response before and after immunization with the seasonal influenza vaccine in adult AML patients who have completed treatment) and 05-H-0206 (Alemtuzumab in Patients with Myelodysplastic Syndrome) which have now completed enrollment and are undergoing analysis and laboratory correlates, we also designed and opened in 2015 a new clinical trial for AML patients with relapsed or refractory disease, PEARL15. Biomarkers for Personalized Early Assessment of Response During Salvage Chemotherapy in People With Relapsed or Refractory Acute Myeloid Leukemia (PEARL15, 15-H-0176, NCT02527447) was presented to the Hematology Branch in April 2015, IRB approved in July, opened in August, and treated the first patient in September 2015. The intent of this trial is to, using our MG-MRD assay as described in the work above, determine if an abnormal peripheral blood gene expression signature on day four of chemotherapy is predictive of failure to achieve complete remission in patients undergoing treatment for relapsed or refractory acute myeloid leukemia. Secondary endpoints will assess if such determination can be made by day 8 of therapy, and if CLIA-grade genetic testing results are also available by day 8 (ie: the potential to select additional therapy based on rational targeting of mutated pathways in subsequent protocols). Target accrual for this protocol is 36 patients over five years. Other clinical trial protocols utilizing novel agents, including in particular immunotherapy, for the prevention and treatment of AML relapse are under development and anticipated in the coming years. Finally, in the laboratory we have begun to develop methodologies with potential utility for high sensitivity monitoring of changes in AML clonality and residual disease burden including next generation sequencing and digital droplet PCR. In an initial proof of principle experiment we were able to identify the acquisition of an additional somatic mutation associated with the rapid progression from MDS to AML in a patient with a germ-line GATA2 mutation. Status: Manuscript in preparation. In summary, the primary interest of the Myeloid Malignancies Section is the detection, prevention and treatment of AML relapse, with the long-term objective of using immunotherapy without the need for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our laboratory work in developing novel methods to detect and quantify AML disease burden with high sensitivity continues, but already provides a foundation to allow correlative assessment of the efficacy of traditional stem cell transplantation and more innovative immunotherapy approaches as tested in clinical trials. In the future we anticipate that the laboratory work of the section will expand in the areas on genomics and digital droplet PCR (detection) paired with a highly translational program of clinical investigation in the prevention and treatment of AML relapse.